Abstract
Article abstract We analyzed genomic DNA from ALS patients for mutations in the apurinic/apyrimidinic endonuclease (APEX nuclease) gene. We identified three rare polymorphisms in the untranslated region of the gene and one common two-allele polymorphism (D148E). The allelic frequency D148E was significantly different in sporadic ALS patients compared with controls. A conserved amino acid change and a 4-base pair deletion were also identified in sporadic ALS patients. These data suggest that APEX nuclease may contribute to the etiology of ALS.
ALS is a degenerative disease of motor neurons, causing progressive muscular atrophy, weakness, and death from respiratory failure. Genetic analysis of ALS patients has identified mutations in the Cu/Zn superoxide dismutase (SOD1) gene in approximately 33% of familial cases and 2.5% of apparently sporadic cases.1,2 Mutations in the SOD1 gene account for such a small proportion of familial cases of ALS that a concerted effort has been made to identify other disease-causing genes.
Defects in DNA repair enzymes have long been postulated as the underlying mechanism responsible for the ALS phenotype.3 Recently, it was reported that levels of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APEX nuclease) were reduced in the frontal cortex of patients with sporadic ALS.4 Sequencing of lymphoblast cDNA from 11 ALS patients and 4 control samples5 suggested that 6 of 11 ALS samples analyzed had mutations in the translated region of the APEX nuclease gene, whereas all 4 control samples were normal, indicating a role for this gene in the etiology of the disease.
The APEX nuclease gene on chromosome 14q11.2-126 is a multifunctional DNA repair enzyme with an important role in the protection of cells from oxidative stress and in the regulation of the DNA-binding of several nuclear factors. The complete gene consists of five exons, of which only two (exons 4 and 5) are translated.
To comprehensively investigate this exciting observation we screened genomic DNA from 117 (SOD1 mutation-negative) sporadic ALS patients, 12 familial ALS patients with known SOD1 mutations, 24 familial ALS patients with no SOD1 mutations, and 58 anonymous control samples for mutations in the APEX nuclease gene.
Methods.
Patients.
Patients analyzed in this study were recruited through the Scottish Motor Neuron Disease Register.7 The anonymous control samples were randomly selected from the healthy Scottish adult population.
Mutation analysis.
Genomic DNA was extracted by standard methods from peripheral blood leukocytes and amplified by PCR using the primers described in table 1. The PCR products were screened for mutations by both single stranded conformation polymorphism (SSCP) and heteroduplex analysis as previously described.8
Primers used in analysis of apurinic/apyrimidic endonuclease (APEX nuclease) mutations
Characterization of mutations.
Samples with aberrant band shifts identified by either method were subsequently sequenced using Thermosequenase 33P terminator cycle sequencing (Amersham Pharmacia Biotech, UK).
Polymorphisms APEX1459 C→T, APEX1835 C→A, APEX2197 T→G, and APEX2698 C→T were checked and confirmed by a modified single nucleotide primer extension protocol (see table 1).9 This method uses cycle sequencing and a primer terminating one base from a mutant single base change to detect the presence or absence of a specific base. The two different polymorphic bases are amplified in separate tests and run adjacently on a polyacrylamide gel to distinguish homozygotes and heterozygotes.
Polymorphism APEX2712 A→T was analyzed by restriction digestion of the relevant PCR product with HinfI and electrophoresis on a 2% agarose gel. Ethidium bromide staining detected a 190-base pair (bp) band in the presence of the common base (A) and 98-bp and 92-bp fragments in the presence of the rare polymorphism (T).
The 4-bp CTCA deletion in exon 4 was confirmed by electrophoresis on an 8% nondenaturing polyacrylamide gel. Two PCR products could be discerned.
The polymorphism 2197 T→G (D148E) was analyzed for linkage disequilibrium by a chi-square test of independence.
Results.
SSCP and heteroduplex analysis of the five exons in the APEX nuclease gene identified six different potential mutations in the samples analyzed. Subsequent sequencing identified two rare polymorphisms in the 5′ untranslated region, one rare polymorphism in the 3′ untranslated region, and one common two-allele polymorphism (D148E) (table 2). D148E variants were analyzed for differences in allele frequency between patients with sporadic ALS and controls and showed a small but significant variation between the two groups of samples (χ2 = 4.877, p = 0.027). There were two other variants: a conserved amino acid change at codon 315 was found in three sporadic ALS patients, and a 4-bp deletion (CTCA) leading to a premature stop codon 174 amino acids from the expected end of the gene was identified in one sporadic ALS patient.
Frequencies of mutations identified in the apurinic/apyrimidic endonulease (APEX nuclease) gene in patients with sporadic ALS compared with anonymous controls
Analysis of patients with familial ALS identified the exon 1 polymorphism 459 C→T in one family with a known SOD1 mutation. The frequencies of the common polymorphism 2197 T→G (D148E) were not significantly different from normal in either SOD1 mutation families (GG 2, GT 8, TT 2) (χ2 = 0.005, p = 0.93) or non-SOD1 mutation families (GG 7, GT 12, TT 5) (χ2 = 0.34, p = 0.55).
Discussion.
We screened genomic DNA from 117 sporadic ALS patients, 12 familial ALS patients with known SOD1 mutations, 24 familial ALS patients with no SOD1 mutations, and 58 anonymous control samples for mutations in the APEX nuclease gene.
We identified three polymorphic base changes occurring in both ALS patients and anonymous normal controls, one single base change present in only two anonymous normal controls, one silent base change in three sporadic ALS patients, and one 4-bp deletion occurring in a single sporadic ALS patient. The 4-bp deletion creates a premature stop at codon 145 predicted to produce a protein 174 amino acids short of the normal product. The major alteration of the protein caused by this deletion could well have a detrimental effect on the function of the enzyme in DNA repair and lead to the observed neurodegenerative disease. The 75-year-old individual with this mutation showed upper and lower motor neuron signs consistent with a diagnosis of ALS. He was abandoned as an infant and has no known family history. Therefore, we cannot determine whether this apparently significant mutation occurred de novo or was inherited, or what role it plays in the etiology of ALS. Unfortunately, this patient died prematurely of coronary heart disease, and no postmortem examination was performed. Therefore, further studies of this mutation are impossible. The silent polymorphism present at Y315 was only identified in three sporadic ALS patients. Whether this polymorphism has any impact on the function of APEX nuclease is unknown.
The common polymorphism D148E was analyzed for allelic segregation, and a significant difference (p = 0.027) between sporadic ALS patients and controls was observed. Patients with familial ALS showed no significant association with either of the polymorphic changes.
The aim of this analysis was to investigate a report of mutations in more than 50% of ALS patients in the translated region of the APEX nuclease gene. The initial study identified mutations in lymphoblastoid cell lines from 6 of 11 ALS patients plus several base changes in the untranslated regions of the gene.5 One of the reported mutations is the polymorphism D148E, which was found in only one patient. Our analysis of genomic DNA derived from peripheral blood leukocytes identified many fewer mutations, with only four sporadic ALS patients having mutations not present in the anonymous controls. The low level of mutations detected in this study suggests a huge discrepancy of mutation rate in the two different populations studied. To eliminate the possibility that mutations were being missed by the SSCP and heteroduplex screening methods, the translated region of APEX nuclease was directly sequenced in a subset of 20 sporadic and 10 familial ALS patients. No further mutations were identified.
In the Scottish cohort of ALS patients, the APEX nuclease mutation rate is less than 3%. This, combined with a significant allelic association with the D148E polymorphism, suggests that the APEX nuclease gene plays a small role in the etiology of ALS but is not a fundamental component in most cases of the disease.
Acknowledgments
Supported by a grant from the Scottish Office Home and Health Department and the Scottish Motor Neurone Disease Association.
- Received January 21, 1999.
- Accepted February 19, 1999.
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